This invention relates in general to methods to monitor the energy enhancing effect of the reduced form of species such as nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), Coenzyme Q10, reduced form of Coenzyme Q10, adenosine triphosphate (ATP) and physiologically acceptable salts thereof, through determination of the relative activity of the energy-producing enzyme NADH cytochrome C reductase in whole blood.
Every living cell needs energy to survive. This energy is produced, according to a process known as oxidative phosphorylation, in form of the chemical entity adenosine triphosphate (ATP). 
The key enzyme in the production of ATP is NADH cytochrome C reductase, also known as Complex I-III. This enzyme reduces cytochrome C by using the reducing agent NADH, the reduced form of nicotinamide adenine dinucleotide. The reduced cytochrome C is then oxidized by the enzyme cytochrome C oxidase (Complex IV) to form water. In other words, the reduced form of NADH, also called Coenzyme I, uses ubiquitous oxygen in the cell to form water and 3 ATP molecules in accordance with the following general reaction scheme:
NADH+H++xc2xdO2+3Pi+3ADPxe2x86x92NAD++3ATP+4H2O.
Thus, with one NADH molecule, three ATP molecules are obtained having an energy of approximately 21 kilocalories. This process is set forth schematically in FIG. 1, where FeS is Reiske iron sulfur protein; ADP=adenosine diphosphate; and b562, b566, c1, a and a3 are cytochromes. The enzymes depicted in FIG. 1 are referred to as Complex I (NADH:ubiquinone oxidoreductase); Complex II (succinate dehydrogenase); Complex III (ubiquinone:cytochrome C oxidoreductase); Complex IV (cytochrome C oxidase); and Complex V (ATP synthase). These enzymes, whose energy-related functions occur in the mitochondria of the cell, are assembled from 13 polypeptides coded by the mitochondrial DNA (mtDNA) and approximately 50 polypeptides coded by the nuclear DNA (nDNA). This system of five complexes also constitutes what is referred to as the electron transport chain (ETC), the common pathway for cellular energy metabolism, through which enzyme-catalyzed redox processes achieve electron transfer among critical substrate species.
NADH cytochrome C reductase is the first and key enzyme of this energy producing process. The greater the activity of NADH cytochrome C reductase, the higher the cellular output of energy. Illustratively, the more energy a cell needs, the more NADH it contains. For example, heart cells have 90 xcexcg/g tissue; brain and muscle cells contain 50 xcexcg/g tissue; liver cells contain 40 xcexcg/g; and red blood cells contain 3 xcexcg/g tissue. Thus, the activity of NADH cytochrome C reductase, directly linked to the amount of NADH present in the cell, reflects the energy producing capacity of a cell. Alberts, B., Bray, D., Lewis, J., Raff, H., Roberts, K., and Watson, J. D., xe2x80x9cEnergy Conversion: Mitochondria and Chloroplasts,xe2x80x9d in Molecular Biology of the Cell, 3rd Ed., Garland Publishing Inc., pp. 653-720, 1994; Lehninger, A. L., xe2x80x9cVitamins and Coenzymes,xe2x80x9d in Biochemistry, 2nd Ed., The John Hopkins University School of Medicine, Worth Publishers, Inc., pp. 337-342, 1975.
It has been shown in a variety of diseases (the so-called mitochondrial diseases) that energy production, in particular the activity of NADH cytochrome C reductase (Complex I-III), is decreased. This has been demonstrated not only in brain and muscle tissue, but also in platelets. Cooper, J. M., Mann, V. N., Krige, D., and Schapira, A. H. V., xe2x80x9cHuman mitochondrial complex I dysfunction,xe2x80x9d Biochemica et Biophysica Acta 1101, 198-203 (1992); Mizuno, Y., et al., xe2x80x9cDeficiencies in Complex I Subunits of the Respiratory Chain in Parkinson""s Disease,xe2x80x9d Biochemical and Biophysical Research Communication 163, 1450-1455 (1989); Shoffner, J. M., Wafts, R. L., Juncos, J. L., Torroni, A., and Wallace, D. C., xe2x80x9cMitochondrial Oxidative Phosphorylation Defects in Parkinson""s Disease,xe2x80x9d Ann. Neurol. 30, 332-339 (1991). This has been found both in patients with Parkinson""s disease (PD) and in patients with Alzheimer""s disease. A further demonstration of the link between cytochrome C reductase activity and disease conditions involves the Parkinson inducing toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a compound that irreversibly inhibits and destroys NADH cytochrome C reductase in certain brain areas causing Parkinsonian-like symptoms. Benecke, R., Strumper, P., and Weiss, H., Brain 1993, Vol. 116, Part 6, pp. 1451-1463. These and other similar findings have significant implications for investigations into the etiology of conditions such as Alzheimer""s and Parkinson""s diseases.
Another known enzyme toxin is azidothymidine (AZT) which is used in the treatment of AIDS patients. This toxin damages NADH cytochrome C reductase, causing a reduction of energy production in the cell. Dalakas, M. C., IIIa, I., Pezeshkpour, G. H., Laukaftis, J. P., Cohen, B, and Griffin, J. L. xe2x80x9cMitochondrial myopathy caused by long-term zidovudine therapy,xe2x80x9d New Engl. J.Med. 322, 1098-1105 (1990). By measuring the activity of NADH cytochrome C reductase in muscle tissue biopsy, it was demonstrated that AZT destroys the enzyme""s activity, consequently blocking the energy production of the cells leading to muscle atrophy.
In addition to substances known to have an inhibitory effect on the activity of key enzymes related to cellular processes for the production of energy, there are also substances, such as the reduced form of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), either endogenous or introduced exogenously, that are able to enhance NADH cytochrome C reductase activity and, consequently, cellular energy production. These two co-enzymes, and their pharmaceutically acceptable salts, have been shown to be useful in the treatment of Parkinson""s Disease (PD). The effectiveness of these agents for this purpose is disclosed in U.S. Pat. Nos. 4,970,200, 5,019,516, and 5,332,727, the disclosures of which are incorporated herein by reference. In addition, these substances are effective in the treatment of Alzheimer""s disease, as disclosed in U.S. Pat. No. 5,444,053, the disclosure of which is also incorporated herein by reference. These substances have also been demonstrated to be effective in supplying additional energy to healthy individuals as disclosed in EP 0 496 479 131.
Assay methods for the determination of NADH cytochrome C reductase activity have been described for many tissues, in particular muscle, liver, brain and heart cells. See, for example, Hatefi, Y. and Stiggall, D. L. (1978b), xe2x80x9cPreparation and properties of NADH:cytochrome C oxidoreductase (Complex II),xe2x80x9d in Methods in Enzymology, 53, Fleischer, S. and Packer, L., eds, pp 5-10, Academic Press, New York, 1978; Trounce, I., Byrne, E. and Marzuki, S., xe2x80x9cDecline in skeletal muscle mitochondrial respiratory chain function: Possible factors in ageing,xe2x80x9d Lancet 1989, 637-639 (assay of muscle tissue); Yen, T.-C., et al., xe2x80x9cLiver mitochondrial respiratory functions decline with age,xe2x80x9d Biochem. Biophys. Res. Comm. 165, 994-1003 (1989) (assay of liver tissue); Nakagawa-Hattori, Y., et al., xe2x80x9cIs Parkinson""s disease a mitochondrial disorder?xe2x80x9d J. Neurol. Sci. 107, 29-33 (1992) assay of muscle tissue obtained post-mortem); Mizuno, Y., et al., xe2x80x9cEffects of 1-methyl-1-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenylpyridinium ion activities of the enzymes in the electron transport system in mouse brain,xe2x80x9d J. Neurochem. 48, 1787-1793 (1987) (assay of mouse brain tissue); Reichman, H. et al., xe2x80x9cRespiratory chain and mitochondrial deoxyribonucleic acid n blood cells from patients with focal and generalized dystonia,xe2x80x9d Movement Disorders 9, 597-600 (1994) (assays of platelet homogenate).
Indeed, cofactors in oxidative phosphorylation processes have seen widespread use in analytical procedures due to the extremely high molar absorptivities demonstrated by species such as NADH which exhibits a unique absorption maximum at approximately 340 nm, along with an even stronger maximum at about 270 nm that is shared with the oxidized form of the cofactor, NAD+. By way of example, the concentration of alcohol in solution can be determined in an enzyme-based assay by adding an excess of NAD+ and a suitable quantity of the NAD+-dependent enzyme, alcohol dehydrogenase. The amount of NADH formed by the enzyme catalyzed reaction, which amount can be easily monitored spectrophotometrically, is stoichiometrically related to the amount of alcohol originally present in solution. The utility of such enzyme-based assays can be further extended through the use of coupled reaction schemes in which the analyte of interest does not directly participate in the enzyme-catalyzed process that gives rise to an analytical signal, but is indirectly linked to such process by a coupled reaction mechanism. However, it should be noted that, despite the use of a common enzyme cofactor in these analytical procedures, all such procedures share a common trait in that they are limited to monitoring processes that are not directly implicated in cellular energy production. As a consequence, such processes are dependent on the addition of both exogenous enzyme and cofactor, such as NADH, in order to generate an analytical signal.
For those demonstrated analytical procedures more directly related to cellular energy processes, a number of specific additional limitations exist. For example, all tissue-based assays require the sampling of tissue through biopsy or other even more invasive surgical procedures. In many cases, the tissue site selected for sampling is so critical physiologically that it can only be sampled post mortem. Assays based on isolation from platelets derived from a blood sample acquired through conventional venipuncture techniques offer advantages over methods that require biopsy of muscle or other tissue. However, such techniques suffer from the serious drawback that observed enzyme activity is dependent on the level of purification of the platelet preparation, introducing a potential source of significant analytical error. Furthermore, platelet separation/purification techniques are inherently complex and time consuming, adding significantly to the procedural overhead of enzyme-based methods from platelets. In addition, all of these enzyme-based assay methods can be further distinguished from the methods of the invention disclosed herein in that they all require the addition of exogenous sources of all cofactors involved in the enzymatic processes. Therefore, none of these methods can be characterized as assessing activity involving endogenous substrate.
In light of these and other significant drawbacks to the currently available assay methods for determination of energy-related enzyme activity, it is recognized that there is a need for an assay method that is simple in both sampling and procedure, as well as one that is efficient and well-suited to routine application. Toward that end, the inventor herein discloses an enzyme-based assay method that can be used with whole blood samples that has utility for assessment of the energy-producing capacity of test subjects, either before and/or after clinical treatments that include the ingestion of exogenous sources of NADH and related compounds.
In one aspect the present invention provides a method for the determination of the relative energy producing capacity of cells present in a sample of whole blood obtained from a test subject, wherein the method comprises the steps of obtaining a sample of whole blood from the test subject, in which sample there is present an endogenous source of an oxidoreductase enzyme participating, directly or indirectly, in cellular energy production; adding to a portion of that sample an effective amount of a substrate associated with the enzyme implicated in cellular energy production; and measurement of an analytical signal the level of which is proportional to the level of an endogenously present cofactor of the enzyme. Preferably, in the method of the invention, the test subject is a mammal. More preferably, the test subject is a human.
In another aspect, the method of the present invention involves an oxidoreductase enzyme selected from the group consisting of NADH:ubiquinone oxidoreductase, succinate dehydrogenase, ubiquinone:cyctochrome c oxidoreductase, cyctochrome C oxidase, ATP synthase, and combinations thereof. Alternatively, the method of the present invention involves an oxidoreductase enzyme selected from the group consisting of cytochrome C reductase (Complex I-III), dihydroubiquinone-cytochrome C oxidoreductase (coenzyme Q10), NADPH cytochrome B reductase, citrate synthetase, isocitrate dehydrogenase, alphaketoglutarate, succinate dehydrogenase, fumarase, and malate dehydrogenase and mixtures thereof.
In still another aspect, the method of the present invention involves an endogenously present cofactor selected from the group consisting of nicotinamide adenine dinucelotide (NAD+), the reduced form of nicotinamide adenine dinucelotide (NADH), nicotinamide adenine dinucelotide phosphate (NADP+), and the reduced form of nicotinamide adenine dinucelotide phosphate (NADPH).
In an alternative embodiment, the present invention contemplates a method for determination of the effect of endogenous factors on the cellular energy producing capability of cells present in a sample of whole blood obtained from a test subject, the method comprising testing a sample of whole blood obtained from the test subject according to a method comprising the steps of obtaining a sample of whole blood from the test subject, in which sample there is present an endogenous source of an oxidoreductase enzyme participating, directly or indirectly, in cellular energy production; adding to a portion of that sample an effective amount of a substrate associated with the enzyme implicated in cellular energy production; and measurement of an analytical signal the level of which is proportional to the endogenous level of an endogenously present cofactor of the enzyme.
In an another embodiment, the present invention provides a method for the determination of the effect of exogenous factors on the cellular energy producing capability of cells present in a sample of whole blood obtained from a test subject, the method comprising the steps of testing a sample of whole blood obtained from the test subject according to a method comprising the steps of obtaining a sample of whole blood from the test subject, in which sample there is present an endogenous source of an oxidoreductase enzyme participating, directly or indirectly, in cellular energy production; adding to a portion of that sample an effective amount of a substrate associated with the enzyme implicated in cellular energy production; and measurement of an analytical signal the level of which is proportional to the endogenous level of an endogenously present cofactor of the enzyme, the sample obtained prior to action of the exogenous factor on the test subject; testing a sample of whole blood obtained from the test subject according to a method comprising the steps of obtaining a sample of whole blood from the test subject, in which sample there is present an endogenous source of an oxidoreductase enzyme participating, directly or indirectly, in cellular energy production; adding to a portion of that sample an effective amount of a substrate associated with the enzyme implicated in cellular energy production; and measurement of an analytical signal the level of which is proportional to the endogenous level of an endogenously present cofactor of the enzyme, the sample obtained after action of the exogenous factor on the test subject; and comparing the results of the first measurement with the second measurement.
Specifically, the method of the present invention encompasses a method wherein the exogenous factor is exposure of the test subject to a substance capable of exerting a toxic effect on an oxidoreductase enzyme participating, directly or indirectly, in cellular energy production, or exposure of the test subject to a substance capable of enhancing the activity of an oxidoreductase enzyme participating, directly or indirectly, in cellular energy production.
In yet another alternative embodiment, the present invention provides a method for the determination of the relative energy producing capacity of cells present in a sample of whole blood obtained from a test subject comprising obtaining a sample of whole blood from the test subject, in which sample there is present an endogenous source of cytochrome C reductase; adding to a portion of that sample an effective amount of cytochrome C; measuring the absorbance of a mixture obtained from the previous two steps at 550 nm 60 seconds after mixing; measuring the absorbance at 550 nm of the mixture of step of the previous step after 300 sec; and calculating the moles of NADH present in the sample according to the following relationship:                               nmol          ⁢                      xe2x80x83                    ⁢                      CytC            red                          =                                                            O                .                D                ⁢                                  .                  550                                ⁢                                  (                                      at                    ⁢                                          xe2x80x83                                        ⁢                    60                    ⁢                                          xe2x80x83                                        ⁢                    sec                                    )                                            -                              O                .                D                ⁢                                  .                  550                                ⁢                                  (                                      at                    ⁢                                          xe2x80x83                                        ⁢                    300                    ⁢                                          xe2x80x83                                        ⁢                    sec                                    )                                                      5                    ·          13                                        =                  nmol          ⁢                      xe2x80x83                    ⁢          NADH          ⁢                      xe2x80x83                    ⁢                      (            endogenous            )                              